Recent developments in the field of the production of biological substances, such as biopharmaceuticals, vaccines and antibodies, have created the need for large-scale manufacturing. Robust and high-yield processes are needed to support the world with sufficient amounts of biological substances to combat all kinds of diseases.
For that reason, great efforts are being put into the optimization of cell-based processes for the production of biological substances. In these processes, cells are being cultured at increasing densities in order to obtain higher product yields per volume. Such high cell density processes are being disclosed in, e.g., WO 2004/099396, WO 2005/095578 or WO 2008/006494. The contents thereof are incorporated herein by reference.
WO 2008/006494 discloses processes for the culturing of cells that produce a biological substance in a bioreactor wherein the cell culture is circulated over a separation system. The separation system separates the cells and the biological substance from substances having a lower molecular weight than the biological substance. The cells and the biological substance are retained together in the reactor and are not separated from each other.
WO 2005/095578 discloses processes for the culturing of cells that produce a biological substance in a bioreactor wherein the cell culture is circulated over a filter module, resulting in an outflow of liquid having a lower cell density than the cell culture and comprising the biological substance. The concentration of cells and biological substance in the outflow are substantially lower than in the bioreactor because the perfusion rate ranges between 1 and 13.7 L/day over a period of 23 days (i.e., the processed volume is 20 times higher than the volume of the bioreactor).
The optimized cell culture processes rely on the ability to culture cells at high cell density (e.g., higher than 10×106 cells/ml) with preservation of a high productivity per cell. Herewith, they offer a method to obtain a harvested solution with high concentration of proteins (e.g., antibodies) in a single bioreactor.
Processes wherein cells are cultured at high densities are prone to the accumulation of high amounts of cell debris and other impurities. These contaminants, together with the cells, have to be discarded further down the purification process, which is a cumbersome operation. As a first step, solid material (such as the cells) and cell debris are to be separated from the cell broth fluid, a step called “clarification.” Examples of clarification methods used to date include centrifugation, filtration (such as microfiltration, depth filtration and filtration through absolute pore size membranes) and expanded bed chromatography.
Many methods for purifying biological substances such as antibodies have been described earlier, e.g., in van Reis et al. (Biotechnology and Bioengineering, 1991, Vol. 38, pp. 413-422). This reference describes a process comprising the steps of recovering cell culture fluid from fermenters and performing a cell-protein separation by tangential flow filtration. This method was, however, only performed with low-density cell cultures comprising about 0.5-4×106 cells/mL. It was not disclosed, nor expected hitherto, that such processes could be applied for a culture containing high cell densities. To the contrary, a strong suggestion could be inferred from the prior art that this process could not be applied on cultures containing high cell densities. In WO 2011/045381, for instance, which relates to the field of virus production, attempts have been made to directly clarify cell cultures having high cell density (see Example 1 of WO 2011/045381). During this attempt, a proper clarification could not be achieved due to rapid clogging of the filter. Apparently, the high cell densities of the broth that was processed impeded the purification step and rendered the tangential flow filtration (TFF) unsuitable for direct clarification. It was only after treatment of the high cell density broth with TRITON® (for lysing the cells), followed by domiphen bromide (DB), that the cell broth could be further processed (by clarification). The crude cell culture was not processed by TFF directly after harvest.
Based on these results, the skilled person would have been discouraged to treat a high cell density harvest directly (without pre-treatment) onto a clarification filter, expecting it to block rapidly.
Methods that are currently in use and considered state of the art for the treatment of high cell density cultures are, for instance, described in WO 2010/043700 or in Schirmer et al. (Bioprocess International, January 2010, pp. 32-39). The latter reference relates to a process wherein the harvest is first diluted, subsequently mixed with Si-PEI beads, after which the beads settle and the supernatant is removed and filtered by depth filtration.
Drawbacks of this method are that the harvest is first diluted 3-5 times depending on the cell concentration in the harvest, which implies that also the antibody titers are diluted 3-5 times. Second, the chromatography resins used in large amounts during the process are very expensive and cannot be re-used. Finally, from experience with this technique, it has been seen that the recovery is variable.
Since cell culture processes are being up-scaled and cells are being cultured at increasing densities, there is a need in the industry for downstream purification processes that enable the treatment of high cell density suspensions, preferably at lower cost, high reliability and increased simplicity as compared to methods described by others. This applies, in particular, to the field of recombinant protein and antibody production.